Main Reagents and Antibodies
Cdk5/p25 protein (active complex) was purchased from Millipore (cat#14-516, Birrica, Massachusetts,USA). The restriction enzyme BamHI, XholI and T4 ligase were purchased from NEB (cat#R0136，cat#R0146，cat#M0202，Ipsich, MA, USA). Competent cells of BL21 and DH5α were from Sangon Bioengineering (cat#B528414，cat#B528413，Shanghai,China). IPTG was from Beyotime Biotechnology (cat#ST098，Shanghai, China). Ni-NTA His-tag purification agarose was purchased from MedChemExpress (cat#HY-K0210，Monmouth Junction, NJ, USA). Anti-phospho-Ser/Thr-proline antibody was obtained from Cell Signaling Technology (cat#9381，Danvers, MA, USA). The antibody against His-tag was purchased from Proteintech (cat#66005-1，Rosemont, IL, USA). Antibodies were used according to the manufacturer’s instructions. All the secondary antibodies (1:10,000) were procured from Jackson ImmunoResearch (Bar Harbor, ME, USA).
Prediction of bioinformatics software
Phosphorylation prediction software GPS5.0 (Group-based Prediction System Version 5 http://gps.biocuckoo.cn.) was used to estimate whether Cdk5 phosphorylating BRCC3, based on the amino acid of mouse-BRCC3 protein (NP_001159929.1). To compare, contrast and analyze the homology and conservatism of BRCC3 in common mammals, DNAMAN software was used to blast the amino acid sequences of human-BRCC3 (NP_001018065.1), mouse-BRCC3 (NP_001159929.1) and rat-BRCC3 (NP_001120772.1) derived from NCBI database.
Construction of pGEX-6P-1-His-BRCC3 plasmid
Primer design and synthesis
Based on gene bank BRCC3 (NM_001166457.1) and vector (pGEX-6P-1) sequences, a pair of primers of BRCC3 with BamHI/Xhol as the restriction sites were designed by Primer Premier 6.0 (BRCC3-F 5'-CGGGATCCATGGCGGTGCAGGTGGT-3' and BRCC3-R 5 '-CCGCTCGAGTTC TAGGGAAGACAGCTCTT -3') and synthesized by Thermo Fisher Scientific (Waltham, MA, USA). The product length is 876bp (full length of BRCC3 gene).
Target gene amplification and plasmid recombination
The plasmid pEGFP-BRCC3 generated previously by our research team was invoked as DNA template, and the target fragment was amplified by RT-PCR. The PCR product was run electrophoresis with 1% agarose gel. The target DNA gel was harvested according to the molecular weight of BRCC3 and extracted with DNA gel extraction kit. In the presence of T4 ligase, the purified DNA product of BRCC3 was ligated at room temperature for 1h with the pGEX-6P-1 vector which digested by BamH I and Xhol I. The ligation product was transformed into DH5α competent cells and monoclonal strains were cultured. The possible recombinant bacterial colony was randomly picked, extracted and then identified as positive colony by double enzyme digestion. The suspected positive plasmid was sent to Sangon Biotech (Shanghai, Co., Ltd) for sequencing to confirm the successful recombination.
Induction and purification of His-BRCC3 fusion protein
The pGEX-6P-1-BRCC3-His construct was transformed into BL21 competent cells, and cultured in LB medium for 14-16 h to observe the growth of the clone. Monoclones were selected and shaken at 37℃ for 16-18 h. Then some bacterial suspension was taken and added into 2×YTA medium (peptone 1.6g, yeast 0.8g, NaCl0.5g, ddH2O supplemented to 100 mL) at a ratio of 1:100, and continued shaking at 37℃ until OD600 to 0.8. At this time, IPTG (0.5 mM) was added into the bacterial suspension and incubated at 37℃. After 4h, the bacterial precipitation was collected by centrifugation. The supernatant was resuspended with lysis buffer (50mM NaH2PO4, 300mM NaCl, 10mM imidazole) and treated with ultrasonic homogenizer. Ni-NTA His-tag purification agarose was used to combine His-BRCC3 target protein according to the manufacture’s instruction, then washed with washing buffer (50mM NaH2PO4, 300mM NaCl, 20mM imidazole), and His-BRCC3 protein was eluted with elution buffer (50mM NaH2PO4, 300mM NaCl, 250mM imidazole). Finally, SDS-PAGE gel was stained with Coomassie brilliant blue R250 to identify the induced His-BRCC3 fusion protein.
Kinase reaction in vitro
For in vitro kinase assays, active Cdk5/p25 complex protein was incubated with purified recombinant His-BRCC3 fusion protein in the kinase reaction buffer (8 mM MOPS/NaOH, 200 nM EDTA) plus 20 μM ATP at 30°C for 30 min. The reaction was stopped by adding SDT buffer (4%(w/v)SDS, 100mM Tris/HCl, 1Mm DTT, pH 7.6). The most of reaction product was analyzed by MALDI-MS/MS. The rest of it was boiled at 95°C for 5 min. The phosphorylation of substrate then was detected by autoradiography with phospho-Ser/Thr-proline antibody (1:1,000) following SDS-PAGE analysis. And total His-tagged proteins were re-probed with anti-His antibody (1:5,000).
Phosphorylation mass spectrometry
The sample of phosphorylated products were analyzed by Shanghai Applied Protein Technology Co. Ltd using LC-MS/MS(Nanolc-QE). Briefly, the chromatographic column was balanced with 95% liquid (solution of 0.1% formic acid), then the sample was loaded from the automatic sampler to the TRAP column for 1 hour. For mass spectral data collection, 20 fragment maps (MS2 scan) were collected after each full scan according to the mass charge ratio of polypeptides. The raw file of mass spectrometry test was retrieved from the database (R20190100160_ZJK.fasta) using Mascot2.2 software, and the identified protein results were obtained.